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RESEARCH ARTICLE
Mo h a m e d A . E l - E s a wi
ABSTRACT:
Brassica napus L. (AACC, 2n = 38) is one of
the most economically important crop spec ies
worldwide. Evaluating the genetic diversity of
this crop species is essential for establishing
efficient conservation and breeding practices.
This study aimed to assess the genetic
diversity, genetic structure and relationships
of 26 Brassica napus accessions of different
origins using SDS-PAGE of total seed
proteins and microsatellite (SSR) markers.
The percentage of protein polymorphism
observed among accessions was 61%. SDSPAGE results also revealed that the total
amount of variation accounted for t he first
three principal components was 73%. Cluster
analysis based on the protein data divided the
26 accessions into 4 groups. The 11 SSR
primer pairs used revealed 14 loci of a total of
50 alleles. The observed heterozygosity
(0.47) was higher than the expected one
(0.298). Polymorphic information content
(PIC) varied from 0.141 to 0.743, with an
average of 0.59 per SSR primer pair . SSR
results also showed that 58.8% of the total
variation was found among accessions, and
41.2% of the SSR variation reside d within
accessions. Cluster analysis based on SSR
data revealed that the two winter oilseed rape
cultivars CGN06870 and CGN17374 were
grouped with the spring oilseed accessions.
The spring oilseed rape cultivar CGN11019
was
grouped
with
the
winter
oilseed
accessions. The genetically diverse spring
and winter oilseed genotypes identied could
be useful resources for oilseed rape breeding.
The useful results of this study could be used
for enhancing and broadening the genetic
base of Brassica napus gene pool.
KEY WORDS:
Brassica
napus,
genetic
diversity,
phylogenetic relationships, SDS-PAGE, SSR.
CORRESPONDENCE:
Mo h a m e d A . E l - E s a wi
INTRODUCTION:
Brassica napus L. (AACC, 2n = 38) is an
important crop species that originated in a
limited geographic region through spontaneous
interspecific hybridizations of Brassica rapa
(AA, 2n = 20) and Brassica oleracea (CC, 2n =
18), followed by chromosome doubling (UN,
1935; Hasan et al., 2006; Fu and Gugel, 2010;
El-Esawi, 2015; El-Esawi et al., 2012). This
species includes oilseed rape, swede or
rutabaga, vegetable types and fodder crops
(Snowdon et al., 2007; W u et al., 2014).
Brassica
napus
is
predominantly
self compatible (McNaughton, 1995; Fu and Gugel,
2010), with interplant out-crossing rates
varying between 20-45% under field conditions
(Olsson, 1960; Rakow and W oods, 1987;
Becker et al., 1992; Damgaard and Loeschcke,
1994). Nowadays, Brassica napus is the
worlds third most important source of
vegetable oil after soybean and palm
(Moghaieb et al., 2014). Plant breeders seek to
develop new Brassica napus cultivars of
nutritionally beneficial high -oleic acid oil to
replace harmful saturated palm oil in food
applications (Spector, 1999; Stoutjesdijk et al.,
2000). In Egypt, the two cultivars of Serw -3
and Serw-4 seem to be promising for the ir high
seed oil contents (40-42%) and should be used
to
increase
Brassica
napus
production
(Moghaieb et al., 2014). However, the limited
geographic range and intensive breeding of
Brassica napus have led to a narrow genetic
basis in its current breeding material (Hasan et
al., 2006; Fu and Gugel, 2010). The gene pool
of Brassica napus breeding material has been
further eroded by an emphasis on oil and seed
quality
traits
(Hasan
et
al.,
2006).
Consequently, genetic variation in Brassica
napus is limited regarding many valuable
characters for breeding purposes. Therefore,
assessment
of
genetic
diversity
and
phylogenetic relationships in this important
crop is essential for establishing efficient
conservation and future breeding practices
(Hasan et al., 2006; Moghaieb et al., 2014).
Over the last years, efforts have been
made to evaluate the genetic diversity and
relationships of Brassica napus germplasm
using a variety of biochemical and molecular
techniques such as total seed proteins using
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El-Esawi MA, Genetic Diversity and Phylogenetic Relationships of Brassica Napus using Protein Profiling and SSR Markers
247
Table 1. Accession numbers, taxonomic group and origin country of the accessions of Brassica napus L. studied.
No.
Accession Number
Country of origin
CGN06892
France
CGN07231
Netherlands
CGN06888
USSR
CGN15178
United Kingdom
CGN06887
New Zealand
CGN19964
Canada
CGN12014
Germany
CGN19968
Netherlands
CGN11019
Poland
10
CGN19967
Sweden
11
CGN06900
Swede (York)
Germany
12
CGN06899
Swede (York)
Germany
13
CGN15779
Netherlands
14
CGN07237
Netherlands
15
CGN06902
Swede
New Zealand
16
CGN06870
Czechoslovakia
17
CGN17305
China
18
CGN17324
Hungary
19
CGN17374
Italy
20
CGN17306
Morocco
21
CGN17308
Ukraine
22
CGN13915
USA
23
Serw-3
Egypt
24
Serw-4
Egypt
25
Semu 249
Egypt
26
Misser L-16
Egypt
SSRs analysis:
A total of 16 SSR primer pairs were
selected from available literatures (Lowe et
al., 2004; Piquemal et al., 2005; Choi et al.,
2007; Long et al., 2007; Rahman and Peter ,
2007; Cheng et al., 2009), and were screened
for
polymorphisms.
Following
the
prescreening, only 11 primer pairs revealed
polymorphism and were used to analyse all
the accessions of the current study. The PCR
reactions were carried out in a final volume of
25 l containing 2 l of genomic DNA (25
ng/l), 1.5 l of forward primer (50 ng/ l), 1.5
l of reverse primer (50 ng/ l), 12.5 l of
GoTaq green master mixture and 7.5 l of
nuclease-free water. The PCR reactions were
then amplified in a Bio -Rad thermocycler
programmed as follows: 94C for 4 min as
initial denaturation followed by 35 cycles of
94C for 45 sec., 55C for 45 sec. and 72C
for 1 min. The PCR products were th en left at
72C for 15 min for final extension. The
amplified PCR products were resolved in 2%
(w/v) agarose gels stained with ethidium
bromide. A 50 bp DNA ladder was used as a
DNA molecular size standard. Bands were
detected and photographed using a UVP gel
documentation system.
Data analysis:
For protein data, molecular weight of
visually clear protein bands was calculated using
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1-F IT = (1-F IS ) (1-F ST ), whereas, F ST is the interaccession genetic differentiation due to genetic
drift, F IS is the fixation index related to nonrandom mating within accessions, and F IT is the
mean inbreeding coefficient of a set of
accessions.
The
polymorphic
information
content (PIC) of each SSR primer pair was also
calculated, as described in Roussel et al.
(2004).
(CGN12014,
CGN19968,
CGN11019,
CGN19967)
and
one
swede
accession
(CGN06902). Group IV consisted of the
remainder of swede accessions (CGN06900,
CGN06899, CGN15779, CGN07237) and one
accession of spring oilseed rape (CGN19964).
RESULTS:
Total seed protein analysis :
Total seed proteins of 26 accessions of
Brassica napus L. were analyzed using
sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE). Table 2 shows
the presence and absence of SDS -PAGE
protein bands in the 26 accessions of
Brassica napus analyzed. In total, 18 protein
bands ranging from 16 kDa to 46 kDa were
observed; 11 bands (61%) were polymorphic
and 7 bands (39%) were monomorphic ( Table
2). Variation was also detected in the density
or sharpness of protein bands.
Table 2. Presence and absence of SDS-PAGE protein
bands in the 26 Brassica napus accessions studied.
Protein
bands
No. of accessions
Molecular
weight (kDa)
Presence
Absence
46
20
43
21
40
26
37
17
35
26
32
12
14
30
14
12
28
26
26
19
10
25
23
11
24
16
10
12
23
16
10
13
22
22
14
22
20
15
21
26
16
20
26
17
17
26
18
16
26
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249
Table 3. Matrix of eigenvectors and values of the principal components for protein data of Brassica napus L. accessions.
Principal components
Accession number
C1
C2
C3
0.606
-0.154
-0.313
0.686
0.160
-0.166
0.826
0.190
-0.212
0.788
0.128
0.063
0.776
0.043
-0.133
0.533
-0.290
0.631
0.717
0.374
0.368
0.749
0.468
0.259
0.395
0.685
0.434
0.666
0.504
-0.059
Swede CGN06900
0.376
-0.609
0.673
Swede CGN06899
0.376
-0.609
0.673
Swede CGN15779
0.495
-0.471
0.575
Swede CGN07237
0.497
-0.367
0.726
Swede CGN06902
0.667
0.341
0.294
0.772
-0.119
-0.367
0.503
-0.302
-0.408
0.781
-0.414
-0.341
0.781
-0.414
-0.341
0.772
-0.119
-0.367
0.781
-0.414
-0.341
0.772
-0.119
-0.367
Serw-3
0.040
0.603
0.084
Serw-4
0.236
0.784
0.178
Semu 249
0.371
0.815
0.050
Misser L-16
0.588
0.263
0.082
10.332
4.844
3.805
39.738
18.630
14.636
Accumulated Eigenvectors
39.738
58.368
73.004
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Table 4. SSR primer pairs, number of alleles and loci, and polymorphic information content (PIC) of each primer set
amplified in the 26 accessions of Brassica napus.
SSR primer pairs
Linkage
group
Sequences
No. of alleles
detected
No. of loci
PIC
CN17
N13
F: CACCATCACCACCTTCACAA
R: TGGTTCACTCATGTCTCCGA
0.743
CN78
N9/N18
F: AGTCGGGCTCGTATATCTCG
R: GTTTCGTGGCGGAAATTAGA
0.560
CB10196
N4
F: TTGTAGGCAATGATGAGGA
R: GAGAGAAGGGCTCCTTTG
0.717
ENA6
N7
F: CTCGTCTTCTTCACCTACAAC
R: CTGACATCTTTCTCACCCAC
0.739
EJU4
N8
F: CACCTTATCATCTCTCTATCCC
R: CCTCTGTTTCTCTCCTTGTG
0.684
Ra2-A01
N7
F: TTCAAAGGATAAGGGCATCG
R: TCTTCTTCTTTTGTTGTCTTCCG
0.597
Niab_ssr013
N5
F: GGAACCGTCCTTACTTTCTCTGT
R: AGGATTGTGTTTTCCACATTGTC
0.563
MR47MR32
SCAR
F: TGAACTGTGGAAGCCAAGC
R: TCACCACTACGCGGTAACTG
0.647
F: GCAGAAGGAGGAGAAGAGTTGG
R: TTGAGCCGTAAAGTTGTCACCT
0.423
BN35D
N1/N11
CNU-SSR149
N6
F: GGAAGCCTCTGTGCGAAAAA
R: TGCCGACGATTTGATAGAGGA
0.689
CNU-SSR223
N3
F: ACCCGAAAAGAGAATATGGCCT
R: ACAGTGGCGTTAGGTGGGG
0.141
50
14
Total
Mean Standard Deviation
4.6 1.2
Genetic
diversity
and
accession -level
heterozygosity:
The genetic diversity of each acces sion
was quantified using the proportion of
polymorphic SSR loci, SSR alle lic count,
heterozygosity, and fixation index (Table 5).
The proportion of polymorphic loci (P) was
variable among accessions, and varied
between 28.6-85.7%, with an average of
51.1%. The mean number of alleles per locus
(A) for each accession varied from 1.286 to
1.857, with an average of 1.51. The effective
number of alleles per locus (A e ) ranged from
1.257 for fodder rape (CGN07231) to 1.857
for winter oilseed rape (CGN06870) , with an
average of 1.49. The observed heterozygosity
(H o ) varied from 0.238 for fodder rape
(CGN07231) to 0.857 for winter oilseed rape
(CGN06870), with an average of 0.47. The
expected heterozygosity (H e ) ranged from
0.162 for fodder rape (CGN07231) to 0.514
for winter oilseed rape (CGN06870 ), with an
average of 0.298. The average fixation
indices values (F) were lower than zero for all
the accessions studied, indicating an excess
of heterozygotes.
Genetic structure:
El e ven ou t of t he 1 4 S SR l oci de tec te d
we re s ta tisti call y si gnif ic an t (p < 0. 00 1 ) f o r
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El-Esawi MA, Genetic Diversity and Phylogenetic Relationships of Brassica Napus using Protein Profiling and SSR Markers
251
Table 5. Percentage of polymorphic SSR loci, estimates of heterozygosity, number of alleles per locus, and
fixation index of the 26 accessions of Brassica napus.
Accessions
Ae
Ho
He
28.6
1.286
1.271
0.262
0.167
-0.569
28.6
1.286
1.257
0.238
0.162
-0.469
28.6
1.286
1.271
0.262
0.167
-0.569
28.6
1.286
1.271
0.262
0.167
-0.569
35.7
1.357
1.343
0.285
0.210
-0.357
64.3
1.643
1.643
0.643
0.386
-0.666
64.3
1.643
1.614
0.595
0.376
-0.583
71.4
1.714
1.671
0.595
0.414
-0.437
35.7
1.357
1.357
0.357
0.214
-0.668
64.3
1.643
1.643
0.643
0.386
-0.666
Swede CGN06900
50.0
1.500
1.471
0.452
0.291
-0.553
Swede CGN06899
50.0
1.500
1.471
0.452
0.291
-0.553
Swede CGN15779
50.0
1.500
1.471
0.452
0.291
-0.553
Swede CGN07237
50.0
1.500
1.457
0.429
0.286
-0.500
Swede CGN06902
57.1
1.571
1.529
0.452
0.329
-0.374
85.7
1.857
1.857
0.857
0.514
-0.667
50.0
1.500
1.500
0.500
0.300
-0.667
50.0
1.500
1.500
0.500
0.300
-0.667
85.7
1.857
1.842
0.833
0.510
-0.633
50.0
1.500
1.500
0.500
0.300
-0.667
57.1
1.571
1.543
0.500
0.316
-0.582
50.0
1.500
1.500
0.500
0.300
-0.667
Serw-3
42.9
1.429
1.429
0.429
0.257
-0.669
Serw-4
42.9
1.429
1.400
0.381
0.248
-0.536
Semu 249
42.9
1.429
1.414
0.405
0.252
-0.607
Misser L-16
64.3
1.643
1.506
0.429
0.302
-0.421
51.1 15.9
1.511 0.16
1.490 0.16
0.470 0.16
0.298 0.09
- 0.578 0.1
P, percentage of polymorphic loci (%); A, the mean number of alleles per locus; A e, the effective number of alleles per locus;
Ho, the observed heterozygosity; He, the expected heterozygosity; F, Wrights fixation index [F = (1 - Ho/ He)].
Table 6. F-statistics and Nei (1973) genetic diversity indices of the 26 accessions of Brassica napus.
Neis genetic diversity indices
F-statistics
X2
0.253
139.7
0.000*
0.074
0.486
83.59
0.000*
0.717
0.330
0.387
97.62
0.000*
0.459
0.739
0.400
0.339
110.5
0.000*
-0.090
0.424
0.684
0.394
0.290
68.71
0.000*
-0.865
0.026
0.478
0.597
0.312
0.285
51.07
0.000*
niab_ssr013
-0.972
0.220
0.604
0.563
0.223
0.340
91.94
0.000*
MR47MR32
-1.000
0.043
0.521
0.647
0.310
0.337
12.88
0.005
BN35D-1
-1.000
0.461
0.730
0.500
0.135
0.365
17.36
0.000*
BN35D-2
-1.000
-0.189
0.405
0.345
0.205
0.140
5.12
0.163
CNU-SSR149-1
-0.699
0.451
0.677
0.723
0.233
0.490
98.34
0.000*
CNU-SSR149-2
0.268
0.902
0.866
0.655
0.088
0.567
133.9
0.000*
CNU-SSR223-1
-1.000
-0.189
0.405
0.345
0.205
0.140
5.12
0.163
CNU-SSR223-1
Mean Standard
Deviation
-1.000
0.754
0.877
0.627
0.077
0.550
104.4
0.000*
-0.886 0.33
0.224 0.39
0.588 0.19
SSR loci
FIS
FIT
FST
HT
HS
DST
CN17
-0.930
-0.275
0.340
0.743
0.490
CN78
-0.801
0.760
0.867
0.560
CB10196
-0.822
0.162
0.540
ENA6
-0.957
-0.060
EJU4
-0.891
Ra2-A01
72.88
F IS , the fixation index related to non-random mating within accessions; F IT , the mean inbreeding coefficient of
a set of accessions; F ST , the inter-accession genetic differentiation due to genetic drift; H T, the total
genetic diversity; H S , the genetic diversity within accessions; D ST , the genetic diversity among
accessions; X 2 , Chi-square value to test F ST for significant difference from zero; and P, the probability
value (*significant F ST at P < 0.001).
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Genetic relationships:
The UPGMA dendrogram constructed
based on SSR data using Euclidean distanc e
matrix on average linkage, showed the
phylogenetic relationships among the 26
accessions of Brassica napus studied (Fig. 2).
It revealed 2 major groups. The first major
group included 2 genetically distinct clusters;
the first distinct cluster contained all swede
accessions,
whereas
the
second
one
comprised 4 accessions of spring oilseed rape
(CGN12014,
CGN19964,
CGN19967,
CGN19968) and only 2 accessions of winter
oilseed rape (CGN06870, CGN17374). The
second major group split into 3 distinct
clusters; the first distinct cluster contained the
four Egyptian cultivars (Serw -3, Serw-4,
Misser L-16 and Semu 249), whereas the
second one included all the accessions of
fodder rape. The third distinct cluster included
4
accessions
of
winter
oilseed
rape
(CGN17305,
CGN17324,
CGN17306,
CGN17308,
CGN13915)
and
only
one
accession of spring oilseed rape ( CGN11019).
Fig.
DISCUSSION:
Evaluation of the genetic diversity and
phylogenetic relationships in crop plants is
essential for establishing efficient conservation
and breeding practices, resulting in developing
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Asghari A, Shokrpour M, Chamanabad HM, Sofalian
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